重组人促红细胞生成素治疗肾间质纤维化的作用机制研究

Mechanism of Recombinant Human Erythropoietin in the Treatment of Renal Interstitial Fibrosis

目的探讨重组人促红细胞生成素（r Hu EPO）治疗肾间质纤维化（RIF）的作用机制。方法 2014年7月—2015年6月,将人肾小管上皮细胞（HK-2细胞）随机分为7组：空白对照组（E1组）：未加任何刺激物;r Hu EPO对照组（E2组）：r Hu EPO终浓度为20 U/ml;人纯化清蛋白诱导组（E3组）：人纯化清蛋白终浓度为5 mg/ml;E4组：加人纯化清蛋白（5 mg/ml）和r Hu EPO（5 U/ml）;E5组：加人纯化清蛋白（5 mg/ml）和r Hu EPO（10 U/ml）;E6组：加人纯化清蛋白（5 mg/ml）和r Hu EPO（20 U/ml）;Rho相关卷曲螺旋形成的蛋白激酶（ROCK）抑制剂Y27632组（E7组）：加ROCK抑制剂Y27632（10μmol/L）,30 min后加人纯化清蛋白（5 mg/ml）。采用细胞增殖试验检测刺激16、24、48、72 h时各组细胞增殖数,反转录-聚合酶链式反应（RT-PCR）法检测Rho A、ROCK mRNA表达水平,细胞免疫荧光法检测α-平滑肌肌动蛋白（α-SMA）、E-钙黏蛋白（E-cadherin）表达水平,酶联免疫吸附试验（ELISA）法检测纤维连接蛋白（FN）表达水平。结果 16 h时,各组细胞增殖数比较,差异无统计学意义（P〉0.05）。24、48、72 h时,各组细胞增殖数比较,差异有统计学意义（P〈0.05）;其中E3-E6组高于E1组、E2组,E4-E6组高于E3组,E7组低于E3组,E5组、E6组高于E4组,E6组高于E5组（P〈0.05）。各组Rho A mRNA表达水平比较,差异有统计学意义（P〈0.05）;其中E3-E6组高于E1组、E2组,E4-E6组低于E3组,E5组、E6组低于E4组,E6组低于E5组（P〈0.05）。各组ROCK mRNA表达水平比较,差异有统计学意义（P〈0.05）;其中E3-E6组高于E1组、E2组,E4-E7组低于E3组,E5组、E6组低于E4组,E6组低于E5组（P〈0.05）。各组α-SMA表达水平比较,差异有统计学意义（P〈0.05）;其中E3-E5组高于E1组、E2组,E4-E7组低于E3组,E5组、E6组低于E4组,E6组低于E5组（P〈0.05）。各组E-cadherin表达水平比较,差异有统计学意义（P〈0.05）;其中E3-E6组低于E1组、E2组,E4-E7组高于E3组,E5组、E6组高于E4组,E6组高于E5组（P〈0.05）。各组FN表达水平比较,差异有统计学意义（P〈0.05）;其中E3-E5组高于E1组、E2组,E4-E7组低于E3组,E5组、E6组低于E4组,E6组低于E5组（P〈0.05）。E3组、E4组、E5组、E6组Rho A mRNA与ROCK mRNA均呈正相关（P〈0.05）。结论人纯化清蛋白能诱导HK-2细胞发生上皮细胞-间充质细胞转分化（EMT）,进而发生RIF,而r Hu EPO通过阻断EMT的Rho A/ROCK信号转导通路从而抑制RIF发生,且在一定范围内r Hu EPO水平越高,其抑制作用越强。

Objective To investigate the mechanism of recombinant human erythropoietin（ r Hu EPO） in the treatment of renal interstitial fibrosis（ RIF）. Methods From July 2014 to June 2015,divided the sampled human renal tubular epithelial cells（ HK-2 cell） into 7 groups： E1 group（ no irritants）, E2 group（ 20 U / ml r Hu EPO）, E3 group（ 5 mg / ml purified albumin）,E4 group（ 5 mg / ml purified albumin ＋ 5 U / ml r Hu EPO）, E5 group（ 5 mg / ml purified albumin ＋ 10 U / ml r Hu EPO）,E6 group（ 5 mg / ml purified albumin ＋ 20 U / ml r Hu EPO）,E7 group（ 5 mg / ml purified albumin 30 min after 10μmol /L Y27632）. At 16,24,48 and 72 h,the number of proliferating cells of each group was detected by cell proliferation assay; Rho A and ROCK mRNA expression levels were detected by RT-PCR; α-SMA and E- cadherin levels were detected by cell immunofluorescence method; fiber connection protein（ FN） expression level was examined by ELISA method. Results At16 h,the 7 groups were not significantly different in the number of proliferating cells（ P〈0. 05）. At 24,48 and 72 h,the 7groups were significantly different in the number of proliferating cells（ P〈0. 05）; E3- E6 groups were higher than E1 group and E2 group; E4- E6 groups were higher than E3 group; E7 group was lower than E3 group; E5 and E6 groups were higher than E4 group; E6 group was higher than E5 group（ P〈0. 05）. The 7 groups were significantly different in the expression level of Rho A mRNA（ P〈0. 05）; E3- E6 groups were higher than E1 group and E2 group; E4- E6 groups were lower than E3 group;E5 and E6 groups were lower than E4 group; E6 group was lower than E5 group（ P〈0. 05）. The 7 groups were significantly different in the expression level of ROCK mRNA（ P〈0. 05）; E3- E6 groups were higher than E1 group and E2 group; E4- E7 groups were lower than E3 group; E5 and E6 groups were lower than E4 group; E6 group was lower than E5 group（ P〈0. 05）.The 7 groups were significantly different in the expression level of α-SMA（ P〈0. 05）; E3- E5 groups were higher than E1 group and E2 group; E4- E7 groups were lower than E3 group; E5- E6 groups were lower than E4 group; E6 group was lower than E5 group（ P〈0. 05）. The 7 groups were significantly different in the expression level of E- cadherin（ P〈0. 05）; E3- E6 groups were lower than E1 group and E2 group; E4- E7 groups were higher than E3 group; E5- E6 groups were higher than E4group; E6 group was higher than E5 group（ P〈0. 05）. The 7 groups were significantly different in the FN expression level（ P 0. 05）; E3- E5 groups were higher than E1 group and E2 group; E4- E7 groups were lower than E3 group; E5- E6 groups were lower than E4 group; E6 group was lower than E5 group（ P〈0. 05）. Rho A mRNA was positively correlated with ROCK mRNA in E3,E4,E5 and E6 group（ P〈0. 05）. Conclusion Purified albumin can induce the occurrence of EMT and further induce RIF. r Hu EPO can inhibit the occurrence of RIF,and its inhibitory effect improves with the increase of r Hu EPO level to some extent.